1. Field of the Invention
The present invention generally relates to methods and kits for isolating and/or detecting IgM antibodies by employing binding substances which are Borrellia burgdorferi cells, or cellular or extracellular components obtained or derived therefrom, as well as to related applications therefor.
2. Description of Related Art
Infection with the spirochete Borrelia burgdorferi causes the acute and chronic manifestations of Lyme borreliosis (Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, and J. P. Davis. 1982. Lyme disease--a tick-borne spirochetosis? Science 216:1317-1319). Despite considerable work on humoral and cell-mediated responses to infection, which has recently been reviewed (Steere, A. C. 1989. Lyme Disease. New. Engl. J. Med. 321:586-596. Szczepanski, A., and J. L. Benach. 1991. Lyme borreliosis: host responses to Borrellia burgdorferi. Microbiol. Rev. 55:21-34), the pathogenic mechanisms that contribute to chronic disease induced by this spirochete remain obscure. It is evident that B. burgdorferi is susceptible to complement-mediated cytolysis and phagocytic clearance (Steere, 1989, pp. 586-596; Szczepanski et al., 1991, pp. 21-34). Furthermore, most, if not all, uncompromised mammalian hosts develop a significant humoral response to infection (Steere, 1989, pp. 586-596; Szczepanski et al., 1991, pp. 21-34). However, the persistent infections documented in humans and animal models indicate that immune clearance is either rare or non-existent (Steere, 1989, pp. 586-596; Szczepanski et al., 1991, pp. 21-34). Several studies have demonstrated passive protection by antibodies, and active protection by vaccination in animal models (Steere, 1989, pp. 586-596; Szczepanski et al., 1991, pp. 21-34). Such protection is apparently transient, requiring immunization during a limited period prior to infectious challenge.
Little evidence suggests that antigenic variation contributes significantly to transient protection and persistent infection (Steere, 1989, pp. 586-596; Szczepanski et al., 1991, pp. 21-34). Although antigenic differences among various isolates have been documented (Barbour, A. G., S. L. Tessier, and S. F. Hayes. 1984. Variation in a major surface protein of Lyme Disease Spirochetes. Infect. Immun. 45:94-100; Schwan, T. G. and W. Burgdorfer. 1987. Antigenic changes of Borrelia burgdorferi as a result of in vitro cultivation. J. Infect. Dis. 156:852-853; Steere, 1989, pp. 586-596; Szczepanski et al., 1991, pp. 21-34), dramatic antigenic changes during chronic infection have not been reported. Furthermore, no evidence for antigenic shifts or gene rearrangements in B. burgdorferi, similar to those believed to function as immune evasion mechanisms in the relapsing fever agent B. hermsii (Barbour, A. H., C. J. Carter, N. Burman, C. S. Freitag, C. F. Garon, and S. Bergstrom. 1991. Tandem insertion sequence-like elements define the expression sites for variable antigen genes of Borrelia hermsii. Infect. Immun. 59:390-397), has been described. Such observations have led to suggestions that B. burgdorferi persistent infections are maintained by spirochetes that become sequestered from immune effectors.
Recent studies using an antigen capture and detection assay demonstrated aggregates of extracellular B. burgdorferi antigens in fluids and tissues from infected arthropod and mammalian hosts (Dorward, D. W., T. G. Schwan, C. F. Garon. 1991. Immune capture and detection of extracellular B. burgdorferi antigens in fluids or tissues of ticks, mice, dogs, and humans. J. Clin. Microbiol. 29:1162-1171). The assay system utilized polyclonal F(ab').sub.2 fragments generated against extracellular vesicle concentrates to capture antigens, which were subsequently labeled and detected using polyclonal IgG antibodies specific for B. burgdorferi. These antibodies were raised against an 83 kDa major extracellular protein (MEP) band, but reacted primarily with OspA and OspB from geographically diverse isolates. The assay enabled consistent detection of the aggregates in samples from which spirochetes were rarely observed, suggesting tat the aggregates were constantly circulating, or that the aggregates were deposited by motile spirochetes and persisted in situ. The study also showed that the antigen aggregates could be recovered from culture supernatants along with extracellular membrane vesicles, and that the aggregates may be derived from s-layer material. However, the apparent relationship between the Osp proteins and the 83 kDa band was not pursued beyond demonstrating the reactivity of anti-MEP IgG antibodies (Dorward et al., 1991, pp. 1162-1171).
IgG-binding proteins have conventionally been used to detect the presence of IgG antibodies in solutions, or on surfaces exposed to IgG antibodies by a variety of techniques. Techniques which are commonly used include: Enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoblot analysis, immunofluorescent assay (IFA), immunohistochemistry, immunoelectron microscopy (IEM), and immunoilluminescence. Each technique utilizes specific protein conjugates to visualize the binding of the protein conjugate to antibody molecules. Two types of proteins are typically used as conjugates. These are bacterial immunoglobulin binding proteins (Fc receptors) and mammalian antibodies directed against other antibodies. Although both types have unique advantages and disadvantages, bacterial proteins generally offer greater stability at less cost per application. Currently, only staphylococcal protein A, and streptococcal protein G, which exclusively bind to IgG antibody molecules are commonly used. A wide variety of enzyme-linked, radioactive, fluorescent, illuminescent, and metallocolloid conjugates of these proteins are commercially available. However, no IgM-specific bacterial protein conjugates have been developed. Therefore, it is desirable to develop an IgM-binding substance or protein which may be used to purify or detect IgM antibodies and which also may be employed in the above-noted applications for IgG-binding proteins.